Mass spectrometry is a technique that analyzes a sample by identifying the mass-to-charge ratio of constituent parts of the sample. Mass spectrometry (MS) has many applications in the study of proteins, known as proteomics. MS may be used to characterize and identify proteins in a sample or to quantify the amount of particular proteins in a sample.
It is known to analyze proteins, peptides or other large molecules in a multistep process. In the example of a protein analysis, in a first portion of the process, the protein may be broken into smaller pieces, such as peptides. Certain of these peptides may be selected for further processing. Because the peptides are ions (or may be ionized by known processes such as electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), or any other suitable process), selection may be performed using an ion trap. The ion trap may be controlled with an oscillating excitation signal. Depending on the frequency of oscillation, ions in the trap of different mass-to-charge ratio (m/z—where m is the mass in atomic mass units and z is the number of elemental charges) will be excited with sufficient energy to escape the trap. What remains in the trap following excitation is ions that did not have a mass-to-charge ratio corresponding to the excitation signal. To isolate ions with a particular mass-to-charge ratio, the ion trap may be excited with a signal that sweeps across a range of frequencies except the frequency that excites the ions of interest. Such an excitation signal is said to have a frequency “notch” corresponding to the target ion that is to be isolated.
The selected ions remaining in the trap may be again broken into smaller pieces, generating smaller ions. These ions may then be further processed. Processing may entail selecting and further breaking up the ions. The number of stages at which ions are selected and then broken down again may define the order of the mass spectrometry analysis, such as MS2 or MS3. Regardless of the order, at the end stage, the mass-to-charge distribution of the ions may be measured, providing data from which properties of the compound under analysis may be inferred. The ions prior to a fragmentation are sometimes called “precursor” ions and the ions resulting from a fragmentation are sometimes called “product ions.” The mass-to-charge distribution may be acquired for any group of product ions. Moreover, all or a subset of product ions from one stage of MS may be used as precursor for a subsequent stage of MS.
This multistep process may be time consuming. It is known to increase the throughput of a mass spectrometry facility by analyzing multiple samples at the same time, which is sometimes referred to as “multiplexing” the samples. The development of specially designed chemical tags, such as tandem mass tags (TMTs) and isobaric tags for relative and absolute quantitation (iTRAQ), has provided the ability to perform multiplexed quantitation of a plurality of samples simultaneously. Performing a multiplexed quantitation allows the relative quantities of particular proteins or peptides between samples to be determined. For example, multiplexed quantitation may be used to identify differences between two tissue samples, which may comprise thousands of unique proteins.
The chemical tags are included in reagents used to treat peptides as part of sample processing. A different tag may be used for each sample. Each of the plurality of tags is isobaric, meaning they have nominally the same mass. This is achieved by using different isotopes of atoms in the creation of the tags. For example, a first tag may use a Carbon-12 atom at a particular location of the molecule, whereas as second tag may use a Carbon-13 atom—resulting in a weight difference of one atomic mass unit at that particular location. This purposeful selection of particular isotopes may be done at a plurality of locations for a plurality of elements. As a whole, each isotope of each tag is selected so that the different types of tags have the same total mass resulting in tagged precursor ions with nominally the same mass despite being labeled with a different type of tag. The different isotopes are strategically distributed within the tag molecule such that the portion of the tag molecule that will become a reporter ion for each type of tag has a different weight. Thus, when the different types of tags are fragmented during the MS analysis techniques, each type of tag will yield reporter ions with distinguishable mass-to-charge (m/z) ratios. The intensity of the reporter ion signal for a given tag is indicative of the amount of the tagged protein or peptide within the sample. Accordingly, multiple samples may be tagged with different tags and simultaneously analyzed to directly compare the difference in the quantity of particular proteins or peptides in each sample.
In the analysis described above, the multi-step processes serves to reduce reporter ion ratio distortion resulting from the fragmentation of co-isolated interfering ions. In particular, if interfering ions tagged with the same type of tag were not completely ejected from the ion trap during the isolation of the target peptides, reporter ions from the tags of the interfering ions may have contributed to the observed signal. In this case, determining the quantity of the tagged target peptide was difficult due to the reporter ions of the target peptides being indistinguishable from the reporter ions of interfering ions. Accordingly, any interfering ion that was co-isolated with the target peptide destroyed the ability to accurately determine the quantity of the target peptide in the sample.
FIG. 1 illustrates this interference problem. In FIG. 1A, a complex mixture of LysC TMT-labeled yeast peptides (ratios 10:4:1:1:4:10) was mixed with a complex mixture of LysC TMT-labeled human peptides (ratios 1:1:1:0:0:0) and analyzed in a series of LC-MS2 and LC-MS2/MS3 based analyses. In FIG. 1B TMT-labeled peptide (NAAWLVFANK—ratios 10:4:1:1:4:10) was interrogated in back-to-back scans using: (1) MS2 scans that fragmented the MS1 precursors using either CID-NCE35 or HCD-NCE45. (2) An MS3 scan that fragmented the MS1 precursor with CID-NCE35, isolated a single MS2 product ion, and then fragmented that ion population using HCD-NCE60. And, (3) an MS3 scan that isolated multiple MS2 product ions, and then fragmented that population using HCD-NCE50.
FIG. 1A shows a complex mixture of LysC TMT-labeled yeast peptides mixes in a one-to-one ratio with a complex mixture of LysC TMT-labeled human peptides. The yeast peptides, for the purposes of this illustrative example, are considered the target and the human peptides generate the interfering ions. The peptide NAAWLVFANK (wherein each letter of the sequence represents an amino acid) of the yeast sample is labeled with one of six TMT tags. The six tags are used in a 10:4:1:1:4:10 ratio and mixed together to make the complex mixture. The human sample is labeled with the six different TMT tags with a 1:1:1:0:0:0 ratio. If there was no interference from the human peptides, the resulting MS spectrum of the tags would perfectly match the original ratio of the target sample, i.e. 10:4:1:1:4:10. This ideal spectrum is illustrated by the MS spectrum on the bottom right of FIG. 1A. However, with interference from the human sample, the MS spectrum is not accurate, as illustrated by the MS spectrum in the top right of FIG. 1A. Due to contributions from the first three tags used to label the peptides of the human sample, the intensity of the peaks associated with the m/z value of the first three tags are not accurate. This interference destroys the ability to accurately determine the relative ratios of each tag used in the yeast sample.
This interference problem is also illustrated in the top spectra of FIG. 1B based on experimental data. The spectrum on the left represents the MS2 product ion spectrum of the above described sample wherein the MS2 precursor ion is fragmented using collision induced dissociation (CID) with a normalized collision energy (NCE) of 35% (CID-NCE35) or high energy beam type dissociation (HCD) with an NCE of 45% (HCD-NCE45). The spectrum on the top right of FIG. 1B represents a portion of the MS2 product ion spectrum showing only the m/z value range from 125-133, which is the range encompassing the m/z values of the six different reporter ions of the six different types of TMT tags used. As discussed above, the ratio of the intensity of the first tag to the third tag should be 10:1 in the absence of interference from the human peptides. In this particular experiment, the ratio is 4.6:1, which is inaccurate by more than a factor of two. This dramatic inaccuracy of the relative quantitation measurement illustrates the need to find a solution to this interference problem caused by co-isolated precursors.